Genetically modified paramyxovirus for treatment of tumor diseases

ABSTRACT

The present invention relates to a genetically modified Paramyxovirus, a pharmaceutical composition comprising this paramyxovirus, the use of a genetically modified Paramyxovirus for the therapeutic and/or prophylactic treatment of a tumor disease, and a method for the production of a pharmaceutical composition for the therapeutic or prophylactic treatment of a tumor disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending international patentapplication PCT/EP2011/056290 filed on 20 Apr. 2011 and designating theU.S., which has been published in German, and claims priority fromGerman patent application DE 10 2010 018 961.8 filed on 23 Apr. 2010.The entire contents of these prior applications are incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains references to amino acid sequences and/ornucleic acid sequences which have been submitted concurrently herewithas the sequence listing text file “5402P453-US_ST25.txt”, file size 41KiloBytes (KB), created on 19 Oct. 2012. The aforementioned sequencelisting is hereby incorporated by reference in its entirety pursuant to37 C.F.R. §1.52(e)(5). The substitute sequence listing in the ASCII textfile entitled “5402P453-US.txt” is hereby incorporated by reference inits entirety. The ASCII text file entitled “5402P453-US.txt” was createdon 4 Dec. 2012 and the size is 45 KB.

FIELD

The present invention relates to a genetically modified Paramyxovirus, apharmaceutical composition comprising this Paramyxovirus, the use of agenetically modified Paramyxovirus for the therapeutic and/orprophylactic treatment of a tumor disease, and a method for theproduction of a pharmaceutical composition for the therapeutic orprophylactic treatment of a tumor disease.

BACKGROUND

Statistically, every third European develops cancer in his lifetime. InGermany every year about 395,000 human beings develop cancer, about195,000 thereof are women and 200,000 are men. Most of these casesdevelop at the age of over 60 years.

Solid tumors are still a big challenge of the clinical oncology. Asignificant improvement of the prognosis of individual tumor diseasescan almost exclusively be reached by establishing new principles oftherapy, integrated into multimodal concepts.

One of these new principles of therapy relates to the application ofreplicating viruses for the treatment of tumors. This approach isreferred to as virotherapy or oncolysis. Numerous viruses have oncolyticproperties with a preferred replication in different tumor cells incomparison to a reduced replication in healthy parenchyma cells.Currently, multiple virotherapeutic agents are subject of severalclinical trials.

Viruses of the family of Paramyxoviridae are of particular interest.Important members of this family of enveloped viruses are the Newcastledisease virus which belongs to the genus of Avulavirus, the Measlesvirus which belongs to the genus of Morbilliviruses, and the Sendaivirus belonging to the genus of the Respiroviruses.

The genome of the Paramyxoviruses comprises a negative single-strandedRNA, i.e. an RNA molecule encoding genes or open reading frames (ORFs)in the anti-sense mode. In a Sendai virus the 3′-head region of the RNAgenome is followed by the viral genes N (Nucleocapsid), P (Phospho), M(Matrix), F (Fusion), HN (Hemagglutinin-Neuraminidase) and L (Large),followed by the 5′-tail region.

The N, P, and L proteins are required for the expression of the genesencoded by the genomic RNA and for the autonomous replication of theRNA. The HN protein supports the infection of specific cell types. Theso-called Matrix protein (M) is a structure protein in the virusparticle which is associated with the membrane.

The F protein has a central function in the infection by inducing thecell membran fusion which is necessary for the initial infection and thevirus expansion to the neighboring cells. It is synthesized invirus-infected cells as an inactive precursor F0 and anchored in thelipid envelope of the virus which originates from the plasma membrane ofthe host cell. F0 is cleaved into the active subunits F1 and F2 by thetryptase “Clara” which can be found in the respiratory tract of rats andmice and is secreted from the bronchial epithelium cells. F1 and F2 havethe capability to fuse cell membranes, thereby initiating the infectionof the host by the virus. Therefore, the cleavage of F0 is a decisivedeterminate for the infectiousness and pathogenity of the Sendai virus.The protease restriction is an important determinant by which theinfection with the Sendai virus in mice is restricted to the respiratorytract and cannot result in a systemic infection.

Kinoh et al. (2004), Generation of a recombinant Sendai virus that isselectively activated and lyses human tumor cells expressing matrixmetalloproteinses, Gene Ther. 11, p. 1137-1145, propose the use of agenetically modified Sendai virus for the treatment of tumor diseases.The principle of the genetic modification is the introduction of anartificial cleavage site into the viral F protein, which is recognizedand can be cleaved by tumor-specific matrix metalloproteinases and,thereby, should enable a tumor-specific replication of the modifiedviruses. Furthermore, the known genetically modified Sendai viruscomprises a deletion in the viral M protein resulting in an inhibitionof the release of offspring viruses in such a way that an expansion ofthe virus is only possible by cell-to-cell contacts via fusion. Thismodified Sendai virus is also disclosed in EP 1 505 154.

Kinoh et al. (2009), Generation of optimized and urokinase-targetedoncolytic Sendai virus vectors applicable for various humanmalignancies, Gene Ther. 16, p. 392-403, reports a genetically modifiedSendai virus having a truncation of amino acids in the viral F proteinwhich should result in an increase of the fusion activity. Furthermore,the viral F protein comprises a so-called “Urokinase Type PlaminogenActivator (uPA) Sensitive Sequence” (SGRS) by which a cleavage andactiviation of F0 through tumor-specific proteases should extend thereplication capacity of the viruses to a multitude of tumors.

Elankumaran et al. (2010), Type I Interferone sensitive recombinantNewcastle-Disease-Virus for oncolytic virotherapy, Journal of Virology,online publication, propose the use of recombinant Newcastle diseaseviruses (rNDV) as an anti-tumor agent which either comprise a mutationin the V protein and is referred to as “rBC-Edit”, or a mutation in theF protein and is referred to as “rLaSota V.F.”.

US 2009/0175826 reports using a recombinant Newcastle disease virus(rNDV) as an oncolytic agent, which comprises a transgene which shouldinduce apoptosis in tumor cell lines.

The oncolytic viruses described in the art so far have not proven ofvalue. A clinical application with a defined proof of effectiveness isstill to be demonstrated. In particular, the oncolytic viruses so farused in the art have the disadvantage of also infecting and destroyingnon-tumor cells to an extended degree. On these grounds the oncolyticviruses used so far are unusable in a clinical application. In addition,it is not clear for which tumor diseases a good effect of individualvirus systems can be reached. For this reason the oncolytic viruses usedso far are not usable in clinical applications.

SUMMARY

Against this background an object underlying the present invention is toprovide an improved oncolytic virus by means of which the disadvantagesof the oncolytic viruses used so far could be largely avoided. Inparticular, such oncolytic viruses should be provided which canreplicate in tumor cells and destroy the latter, however which replicatein non-tumor cells only in a strongly restricted fashion and, thereby,fulfill a sufficient epidemiological safety aspect.

This object is achieved by the provision of a genetically modifiedParamyxovirus, preferably a genetically modified Sendai virus (SeV)which, in reference to the wild type (wt), comprises in its F gene atleast a first genetic modification and in its P gene at least a secondgenetic modification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic structure of the Sendai virus.

FIG. 2 shows the structure of the pSVV10 plasmide with the cDNA of theSendai virus.

FIG. 3 shows the open reading frames (ORFs) of the non-structuralaccessory genes in the P gene.

FIG. 4 shows the subcloning into the cloning vector pSL1180.

FIG. 5 shows the principle of the mutagenesis PCR.

FIG. 6 shows the primer procedure for the verification of the mutationsafter the mutagenesis PCR.

FIG. 7 shows step 1 of the recloning where the mutated SphI-EcoRIfragments were excised from the pLS1180 Sph1-Eco pSVV10 cloning vectorand cloned into a Sendai virus vector pVV13.

FIG. 8 shows step 2 of the recloning; into the pSVV13 vector lacking ofthe ORFs for M, F, HN; this region has been cloned with the amended Fcleavage site from pRS Id-E Fm via EcoRI; the resulting vectors containthe mutated P/C region and a ubiquitous F cleavage site from NDV.

FIG. 9 shows the replication of viruses according to an embodiment ofthe invention on vero producer cells and hepatoma cells (Hep3B, HuH7,PLC/PRF/5).

FIG. 10 shows a replication of the viruses according to an embodiment ofthe invention on non-tumor cells (MRC-5, human fibroblasts; PHH, primaryhuman heptocytes).

FIG. 11 shows the amplification of the viruses according to anembodiment of the invention in vivo.

DETAILED DESCRIPTION

As used herein, “Paramyxovirus” refers to such a virus which belongs tothe family of Paramyxoviridae and a virus resulting therefrom by the wayof propagation. Paramyxoviruses encompass the subfamily ofParamyxovirinae with the genera of Respiroviruses, Rubulaviruses,Avulaviruses, and Morbilliviruses as well as the subfamily ofPneumovirinae with the genera of Pneumoviruses, and Metapneumoviruses.Details on the taxonomy of the Paramyxoviruses can be found, for examplein Kneipe et al. (2007), Fields Virology, 5th Edition, LippincotWilliams & Wilkins.

As the inventors were able to find out, basically every strain of theSendai virus is qualified for the modification according to theinvention. Especially suitable strains of the Sendai virus are“Fushimi”, “Harris”, “Z-Strain”, “Ohita”, “Hamamatsu”, “Cantell”, and“52”.

The inventors have realized for the first time that a geneticmodification of the F gene or the F protein, respectively, and theabolishment of the protease restriction provides for an efficientexpansion of the virus in the tumor tissue so that a broad spectrum ofdifferent tumors can be infected.

The nucleotide sequence of the F gene of the Sendai virus is depicted inthe enclosed sequence listing under SEQ ID NO: 1 and has the GeneID1489775. The amino acid sequence of the F protein of the Sendai virus isdepicted in the enclosed sequence listing under SEQ ID NO: 2 and has thedatabase access nos. BAA 24390.1 or NP_(—)056877.

The inventors have also realized that the second genetic modification inthe P gene or P protein, respectively, results in an attenuation of thevirus. Because of the genetic modification or mutation in the P gene,respectively, the reading frames for the genes or proteins C′, C, Y1, Y2are shifted. However, the P protein remains completely intact.

The nucleotide sequence of the P gene of the Sendai virus is depicted inthe enclosed sequence listing under SEQ ID NO: 3. The amino acidsequence of the P protein is depicted in the enclosed sequence listingunder SEQ ID NO: 4 and has the database access nos. BAA 24386.1 and AAB06279.

The P gene encodes for several accessory non-structural proteinsreferred to as C′, C, Y1, Y2, V and W. These proteins are generated byoverlapping reading frames and “RNA editing”. The exact functions of theaccessory non-structural proteins are not fully illucidated in detail,whereas it is assumed that they can interact with the unspecific defensesystem of the infected cells, such as the interferon system.

The Paramyxoviruses as modified according to the invention are, incontrast to the wild type viruses, in the position to independentlyreplicate in the tumor tissue and to destroy the latter, whereas norestriction to a specific tumor cell type can be observed. Surprisingly,the virus according to the invention can only replicate to a verylimited extend in non-tumor cells, such as primary human hepatocytes orfibroblasts. As a result, the virus according to the invention infectstumor cells with a very high preference in reference to nontumor cells.Damages of normal tissue are significantly reduced whereas thetherapeutic scope of the virus is increased.

As used herein “genetic modification” refers to a preferably targetedalteration of the genome, the genetic information or the encodedproteins of the Paramyxovirus of the invention in reference to the wildtype, which can e.g. be introduced by targeted mutagenesis.

In one embodiment the first genetic modification is designed in such amanner that it results in a tropism extension.

This measure has the advantage that the virus according to the inventionbecomes independent from specific proteases which e.g. are onlyexpressed by a small number of tissues or tumor cells. In this context,for example the wild type Sendai virus depends on a tryptase which isonly expressed by bronchial epithelium cells and, for this reason, itcan only infect these cells. The virus described by Kinoh et al. (2009;I.c.) depends on the matrix metalloproteinase (MMP) and, therefore, canonly infect such tumor cells which express MMP. In contrast, the furtherdeveloped virus according to the invention is in the position to infectand lyse all tumor cells by reason of the first genetic modification inthe F gene.

In another embodiment the second genetic modification is configured toresult in a tropism restriction.

This measure has the advantage that the virus safety is increased andside effects are significantly reduced. The virus according to theinvention selectively lyses tumor cells, however not healthy cells oronly to a very limited extend. The inventors have realized that throughthe targeted genetic modification of a P gene, i.e. a non-structureprotein, the mode of response of the cell to be lysed can be influencedin such a way that the virus replicates in tumor cells in a targetedmanner. This approach differs from those described by Kinoh et al.(2009; I.c.) where a deletion of the M gene which encodes a structureprotein, results in the synthesis of an incomplete virus particle. Thatalso results in the reduction of the lysis activity. Surprisingly, thelysis activity of the virus according to the invention is, restricted totumor cells, very high.

It is preferred if through the first genetic modification in the F genethat the nucleotide sequence encoding for the SeV WT protease cleavagesite, which comprises the amino acid sequence VPQSR (SEQ ID NO: 5), isreplaced by a nucleotide sequence encoding for a ubiquitous proteasecleavage site, preferably from the F gene of the Newcastle disease virus(NDV), which comprises the amino acid sequence RRQKR (SEQ ID NO: 6).

The modification of the proteolytic SeV-WT protease cleavage site in theF protein according to the invention abolishes the protease restrictionof the wild type. The F protein can then be cleaved by ubiquitousproteases by which an independency from specific proteases which mightonly be present in the respiratory tract of mice or also fromtumor-reducing proteases can be reached. As a result, the geneticallymodified virus can be used for a broader spectrum of tumors, whereas therisk of the development of a resistance is significantly reduced.

In another embodiment the second genetic modification in the P generesults in a modification of an accessory non-structural protein encodedby the P gene, preferably in such a way that at least one of the latteris not transcribable, further preferred not transcribable because of afunctional destruction of the start codon.

These measures have the advantage that the virus as modified accordingto the invention is attenuated in such a way that the replication innon-malign cells and the infection of healthy tissue is significantlyrestricted. Consequently the viruses according to the invention infecttumor cells in a more targeted manner and are capable to lyse them.

It is preferred if the accessory non-structural proteins are selectedfrom the group consisting of: C′, C, Y1, Y2, V and W, wherein it ispreferred if through the second genetic modification in the P gene atleast the C′/C and Y1/Y2 proteins, further preferred at least the C′/C,V, and W proteins, further preferred at least the C′/C, V, W and Y1/Y2proteins are modified or functionally destructed, respectively.

As the inventors have been able to find out in a model system viruseswhich are modified in such a manner comprise a particularly highselectivity for tumor cells and a particularly poor replication capacityin non-tumor cells.

The nucleotide sequence of the C′ gene of the Sendai virus is depictedin the enclosed sequence listing under SEQ ID NO: 7 and comprises theGeneID AB005796.1. The amino acid sequence of the C′ protein of theSendai virus is depicted in the enclosed sequence listing under SEQ IDNO: 8 and comprises the database access number BAA 24394.

The nucleotide sequence of the C gene of the Sendai virus is depicted inthe enclosed sequence listing under SEQ ID NO: 9. The amino acidsequence of the C protein of the Sendai virus is depicted in theenclosed sequence listing under SEQ ID NO: 10 and has the databaseaccess number BAA 24396.

The nucleotide sequence of the V gene of the Sendai virus is depicted inthe enclosed sequence listing under SEQ ID NO: 11. The amino acidsequence of the V protein of the Sendai virus is depicted in theenclosed sequence listing under SEQ ID NO: 12 and comprises the databaseaccess number BAA 20021.

The nucleotide sequence of the W gene of the Sendai virus is depicted inthe enclosed sequence listing under SEQ ID NO: 13. The amino acidsequence of the W protein of the Sendai virus is depicted in theenclosed sequence listing under SEQ ID NO:14 and comprises the databaseaccess number AAX07444.

The nucleotide sequence of the Y1 gene of the Sendai virus is depictedin the enclosed sequence listing under SEQ ID NO: 15. The amino acidsequence of the Y1 protein of the Sendai virus is depicted in theenclosed sequence listing under SEQ ID NO: 16 and has the databaseaccess number BAA 24388.

The nucleotide sequence of the Y2 gene of the Sendai virus is depictedin the enclosed sequence listing under SEQ ID NO: 17. The amino acidsequence of the Y1 protein of the Sendai virus is depicted in theenclosed sequence listing under SEQ ID NO: 18 and has the data baseaccess number AAX07449.

In another embodiment the virus according to the invention comprises, inreference to the wild type (wt), at least one transgene, preferably asuicide gene or other cell death inducing or immunostimulating genes.

This measure has the advantage that the cytotoxicity of the virusaccording to the invention is again increased as the product of thetransgene additionally contributes to an intensified destruction of theinfected tumor cells. Transgenes having an anti-tumor effect are alsoencompassed.

Against this background a further embodiment of the present invention isa pharmaceutical composition comprising the genetically modifiedParamyxovirus according to the invention and a pharmaceuticallyacceptable carrier.

Pharmaceutically acceptable carriers are comprehensively described inthe state of the art, for example in Bauer et al. (1999), Lehrbuch derPharmazeutischen Technologie, Wissenschaftliche Verlagsgesellschaft mbHStuttgart, Kibbe et al. (2000), Handbook of Pharmaceutical Excipients,American Pharmaceutical Association and Pharmaceutical Press. Thecontent of the before-mentioned publications is subject of the presentdisclosure. It goes without saying that the pharmaceutical compositionmay comprise further accessory and active agents, such as cytostatics.

Another embodiment of the present invention is the use of thegenetically modified paramyxovirus according to the invention for thetherapeutic and/or prophylactic treatment of a tumor disease, preferablythe development of a solid tumor, further preferred the development of ahepatocellular carcinoma.

The hepatocellular carcinoma has an incidence of one million each yearand thus is worldwide one of the most frequent malignomas. Only in fewcases a curative therapy is possible by means of resection and livertransplantation. So far convincing alternative concepts of therapy arelacking, since a distinct resistance against all chemotherapeuticstested so far can be observed. These needs are effectively met by theinvention.

Another embodiment of the present invention is a method for theproduction of a pharmaceutical composition for the therapeutic and/orprophylactic treatment of a tumor disease, preferably the development ofa solid tumor, further preferred the development of a hepatocellularcarcinoma, comprising the following steps: (1) Providing the geneticallymodified Paramyxovirus according to the invention, and (2) formulatingthe genetically modified Paramyxovirus into a pharmaceuticallyacceptable carrier.

It is to be understood that the features mentioned before and those tobe explained in the following cannot only be used in the combination asspecifically indicated but also in other combinations or in isolatedmanner without departing from the scope of the invention.

The present invention will now be explained by means of embodimentsresulting in further features, advantages and characteristics of theinvention. Reference is made to the enclosed figures.

EXAMPLES 1. Genomic Organisation of the Sendai Viruses

Sendai viruses are negative strand RNA viruses with a genome ofapproximately 15 kb. The genomic organization is shown in FIG. 1. Thegenes of the six structure proteins are arranged on the viral genomicRNA having the order of 3′-N-P-M-F-HN-L-5′. In front of the first genethe leader region (Id) encompassing 54 nucleotides is located and thelast gene is followed by the trailer region (tr) having a length of 57nucleotides. The numbers below each gene correspond to the gene lengthin bp. For the transcription of an individual gene the viral polymerasestarts with the synthesis of the mRNA at a conserved sequence at the 3′end referred to as gene-start-motive (GS), followed by a untranslatedregion (UTR), the open reading frame (ORF) of the viral gene, andfinally the gene-end-motive (GE) where the mRNA synthesis stops. Betweentwo expression units a conserved intergenic motiv (I) of 3 by can befound which is not incorporated into the mRNA.

The basis of the invention is provided in form of the so-called pSVV10plasmid, more precisely the pSVV10IdGFPMFHN plasmid with a size of19.774 by which encodes the cDNA of the Sendai virus of the strainFushimi, ATCC VR-105. The plasmid is depicted in FIG. 2.

2. Detailed Illustration of the Accessory Proteins Encoded by the P Gene

The accessory proteins C′, C, Y1, Y2, V and W of the Sendai virus areencoded by the P gene. The ORFs of the C and the Y proteins are shiftedby +1 base. The V and W proteins are generated according to the numberof the inserted G proteins at the so-called editing site (ES). The P-ORFsequence region with the accessory genes is shown in FIG. 3 where thenumbers refer to the specifications in the PSVV10 vector.

3. Subcloning in the Mutagenesis Plasmid

To insert point mutations into the genes of the accessory proteins amutagenesis PCR was performed. However, for this method only plasmidshaving a maximum size of 8 kb can be used. The P/V/C region of thegenome of the Sendai virus was subcloned from the Sendai virus vectorpSVV10 (19774 bp) via the restriction enzymes EcoRI and SphI into thecloning vector pSL1180 (3422 bp). For this, both of the vectors ofpSVV10 (FIG. 2) and pSL1180 were digested with the enzymes EcoRI andSphI and the region of pSVV10 (2254 bp) to be mutated was ligated to thevector backbone of sPL1180 (3303 bp). The resulting cloning plasmidpSL1180 SphI-Eco pSVV10 has a size of 5557 bp; cf. FIG. 4.

4. Mutagenesis

By means of targeted mutation the transcription of the accessory genesC′, C, Y1 and Y2 should be prevented. A mutation in the editing site hasalso the effect that the editing can no longer happen. The followingoverview shows the relevant cloning area of pSL1180 with all gene startsand the editing site (SEQ ID NO: 19):

GTCCTGTCGAGTGAACCAACTGACATCGGAGGGGACAGAAGCTGGCTCCACAACACCATCAACACTCCCCAAGGACCAGGCTCTGCCCATAGAGCCAAAAGTGAGGGCGAAGGAGAAGTCTCAACACCGTCGACCCAAGATAATCGATCAGGTGAGGAGAGTAGAGTCTCTGGGAGAACAAGCAAGCCAGAGGCAGAAGCACATGCTGGAAACCTTGATAAACAAAATATACACCGGGCCTTTGGGGGAAGAACTGGTACAAACTCTGTATCTCAGGATCTGGGCGATGGAGGAGACTCCGGAATCCTTGAAAATCCTCCAAATGAGAGAGGATATCCGAGATCAGGTATTGAAGATGAAAACAGAGAGATGGCTGCGCACCCTGATAAGAGGGGAGAAGACCAAGCTGAAGGACTTCCAGAAGAGGTACGAGGAGGTACATCCCTACCTGATGAAGGAGAAGGTGGAGCAAGTAATAATGGAAGAAGCATGGAGCCTGGCAGCTCACATAGTGC

AAAAGAAGACCTACCAACAGTGGGTCCAAACCTCTTACTCCAGCAACCGTGCCTGGCACCCGGTCCCCACCGCTGAATCGTTACAACAGCACAGGGTCACCACCAGGAAAACCCCCATCTACACAGGATGAGCACATCAACTCTGGGGACACCCCCGCCGTCAGGGTCAAAGACCGGAAACCACCAATAGGGAC

TTGGTGTAATCCAGTCTGCTCAAGAATTCGAGTCATCCCGAGACGCGAGTTATGTGTTTGCAAGACGTGCCCTAAAGTCTGCAAACTATGCAGAGATGACATTCAATGTATGCGGCCTGATCCTTTCTGCCGAGAAATCTTCCGCTCGTAAGGTAGATGAGAACAAACAACTGCTCAAACAGATCCAAGAGAGCGTGGAATCATTCCGGGATATTTACAAGAGATTCTCTGAGTATCAGAAAGAACAGAACTCATTGCTGATGTCCAACCTATCTACACTTCATATCATCACAGATAGAGGTGGCAAGACTGACAACACAGACTCCCTTACAAGGTCCCCCTCCGTTTTTGCAAAATCAAAAGAGAACAAGACTAAGGCTACCAGGTTTGACCCATCTATGGAGACCCTAGAAGATATGAAGTACAAACCGGACCTAATCCGAGAGGATGAATTTAGAGATGAGATCCGCAACCCGGTGTACCAAGAGAGGGACACAGAACCCAGGGCCTCAAACGCATCACGCCTCCTCCCCTCCAAAGAGAAGCCCACAATGCACTCTCTCAGGCTCGTCATAGAGAGCAGTCCCCTAAGCAGAGCTGAGAAAGCAGCATATGTGAAATCATTATCCAAGTGCAAGACAGACCAAGAGGTTAAGGCAGTCATGGAACTCGTAGAAGAGGACATAGAGTCACTGACCAACTAG

 reading frame of P/W/V + 1 ATG = Start P/W/V 

 reading frame 1

 reading frame of P/W/V + 1

 reading frame of P/W/V + 1

 reading frame of P/W/V + 1

 

Three specific primers were developed which, on one side, functionallydestruct the start sequences of the genes for the accessory proteins, onthe other side which do not result in a change of an amino acid in the Preading frame.

With the primers in the mutagenesis PCR the following mutations havebeen generated: Desired mutations:

C and C Y1 and Y2 Editing site knock-out knock-out: knock-out: 4308 T→C4377 T→C 5241 A → G C-START_(ko) Y1-START_(ko) 4320 T→A 4395 T→C 5244 G→ A {close oversize bracket} Editing Site_(ko) C-STOP Y2-START_(ko) 4338T→A 5253 A → T C-STOP

The “QuickChange Multi Site directed Kit” of the company Stratagene®enables a targeted insert of point mutations for plasmids of a size ofup to 8 kb in three succeeding steps. In the first step the mismatchedprimers comprising individual point mutations aneal to the denaturatedtemplate single strand when given to the reaction. It has to be takencare that all primers bind to the same template strand. The PfuTurbopolymerase, beginning at the primers, extends the complementary sequencewithout displacing the primers. The newly generated DNA strand nowincorporates the mutation and single overhanging ends, so-called“nicks”, which are adequately displaced by components of the enzymeBlends.

In the second step a digestion with Dpnl is made resulting in adigestion of specifically methylized and hemimethylized DNA. Sinceplasmids which have been amplified in Escherichia coli are dammethylized only the parental template is digested however not the copiesgenerated in the PCR, which contain the mutations.

In the last step the ssDNA is transformed in XL10 gold ultra-competentcells and there converted into dsDNA in vivo. Now the plasmids can beisolated from the bacteria and analyzed for the inserted mutation bymeans of sequencing. The principle of the mutagenesis PCR is depicted inFIG. 5.

The mutagenesis assay is made up as follows (Tab. 1):

TABLE 1 Assay of the SeV mutagenesis PCR for the generation ofrecombinant Sendai viruses with partial deletions in the accessoryproteins Components Vko Cko/Yko Cko/Yko/Vko 10 x Quick Change Puffer 2.5μl 2.5 μl 2.5 μl Quick Solution 0.75 μl 0.75 μl 0.75 μl dNTP Mix 1 μl 1μl 1 μl QuickChange Mutli Enzyme 1 μl 1 μl 1 μl Blend Plasmid 100 ng 100ng 100 ng Primer SeV V_(ko) 0.9 μl — 0.9 μl Primer SeV C_(ko) — 0.9 μl0.9 μl Primer SeV Y_(ko) — 0.9 μl 0.9 μl H₂O ad 25 μl ad 25 μl ad 25 μl

The mutagenesis PCR resembles a conventional PCR, only the extensiontime is very long since the complete vector has to be complemented and,therefore, it varies for each vector: Two minutes per kb; here:pSL1180+Eco-SphI fragment from pSVV10˜5.5 kb corresponds to 11 minutesextension; cf. Tab. 2:

TABLE 2 Mutagenesis PCR Program Mutagenesis PCR Polymerase activation95° C. 01:00 min Denaturation 95° C. 01:00 min Annealing 55° C. 01:00min Extension (depending on 65° C. 11:00 min {close oversize bracket} 35cycles Template) Extension 72° C. 10:00 min  4° C. ∞

Directly after the amplification 1 μl of Dpnl restriction enzyme (10U/μl) is given to the PCR assay, resuspended with the pipette, shortlycentrifuged and digested for one hour at 37° C. The resulting ssDNA wastransformed into XL10 gold ultra-competent cells and isolated from thebacterial as dsDNA-mutated plasmid by means of Miniprep; cf. Tab. 3:

TABLE 3 Sequence modified by mutagenesis; C START C START C-STOP C-STOPWT //ACG-// -/-ATG-/- -/-TTA-/- -/-TTG-// C' and C (-) -C- -A- -A-Kurotani* -A- -A- Gotoh^($) G-- -A- -A- Y1 START Y2 START WT//ATGTTA-//-------------- /-ATG-// Y1 and Y2 (-) - C - -C- Kurotani*- C - - A - -C- Gotoh^($) - C - - A - -C- Editing site ko STOP STOP WT//ACAAAAAAG GGC ATA GGA GAG Editing site --G - - A - - T +1G -TGA- +2G-TGA- Kurotani* Gotoh^($) --G - - A *Kurutani et al. Genes to Cells,1998 ^($)Gotoh et al. FEBS letters, 1999

5. Sequencing

The resulting mutated plasmids were verified for the correct insert ofthe mutations. The primer procedure with the inclusion of the entiremutation region is shown in FIG. 6. The upper numbers refer toinformation for positions in the original vector psVV10 (˜19 kb), thelower numbers refer to information for positions in the pSL1180+pSVV10cloning vector (˜5.5 kb).

6. Recloning of the Mutated Sequences into a Vector Encoding for theComplete Sendai Virus

The mutated sequence region had to be recloned back into a vector withthe complete cDNA sequence of the Sendai virus. Since viruses are to beproduced which should have a ubiquitous F cleavage site instead of acleavage site only activatable by the tryptase “Clara” in therespiratory tract of rodents the eco-eco region of the vector pRSId-EGFP Fmut (19958 bp, Sascha Bossow, MPI Munich) has been used.Instead of the cleavage site in the F protein of the Sendai virus withthe nucleotide sequence (GTTCCACAGTCGAGA; SEQ ID NO: 33) it incorporatesa ubiquitous cleavage site of the F protein from the Newcastle diseasevirus with the sequence (CGTCGTCAGAAGAGA; SEQ ID NO: 34).

At first the different mutated SphI-EcoRI fragments were excised fromthe pSL1180 Sph1-Eco pSVV10 cloning vector and cloned into a Sendaivirus vector pSVV13 which is similar to the vector pSVV10, however lackof the regions for the F gene and the HN gene. Then into such vector thelacking regions for the F gene and the HN gene were cloned with themodified cleavage site in the F gene from pRS Id-EGFP Fut via EcoR I.The resulting vectors are referred to as pSeVmut. Functional Sendaiviruses were produced by the “rescue” method via the transfection ofBSR-T7 cells. Step 1 of the recloning is shown in FIG. 7, and step 2 ofthe recloning is shown in FIG. 8.

7. Production of Recombinant Sendai Viruses

By the “rescue” method for Paramyxoviridae it is possible to producegenetically modified viruses. For this BSR-T7 cells which constitutivelyexpress the T7 RNA polymerase were co-transfected by lipofection withhelper plasmids and plasmids encoding the cDNA of the Sendai virus. Theplasmids controlled by a T7 promoter are transcribed in the cellresulting, in several steps, in viral proteins and viral negative strandRNA genomes and in the generation of functional viruses.

BSR-T7 cells (3×10⁵ per cavity of a plate with six cavities) are seededand cultivated overnight at 37° C. For the transfection 200 μl of DMEMwith FuGENE 6 are put into a vial (the amount of Fugene 6 correspondedto a ratio of 2 μl per μg of DNA) and were incubated for five minutes.After the addition of the DNA components which are listed in the Tab. 4another incubation step is performed for 25 minutes at room temperature.

TABLE 4 DNA components of a “rescue” assay Component Amount of DNA SeVcDNA 7.5 μg pTM-N 250 ng pTM-P/C⁻ 150 ng pTM-L 50 ng

The BSR-T7 cells were washed for two times with DMEM; in the following1.8 ml of DMEM medium+2% FCS is provided. The incubated transfectionmixture was added dropwise under agitation and the cells were incubatedfor three days at 33° C. Thereafter the transfected BSR-T7 cells werewashed for three times each with 1 ml DMEM to remove plasmid remainders.1 ml of fresh DMEM medium+2% FCS was added to each assay and the newlygenerated viruses were harvested after one day.

Before the transfer to Vero cells for the amplification the viruscontaining supernatant was centrifuged at 300 rpm for four minutes atroom temperature. Vero cells which were prepared four days before(plated: 2×10⁵ cells per 3.5 cm dish; set: approx. 10⁶ cells per 3.5 cmdish at the day of the infection) were two times washed with DMEM andinfected with 100 to 500 μl of BSR-T7 supernatant (ad 500 μl adsorptionvolume with culture medium) for one hour at 33° C. under agitation(every 15 minutes). The inoculum was removed, the cells were washed fortwo times with DMEM and incubated in 1 ml culture medium for two to fivedays under daily exchange of medium at 33° C. As soon as eGPF wasdetectable as viral encoded marker protein in the fluorescencemicroscope the culture supernatant was removed. With this initial viruspassage further passages (passages two and three) were produced at alarger scale. The titer of the virus offspring was quantified by theTCID₅₀ method.

The following genetically modified or recombinant Sendai virusesaccording to the invention were produced:

-   -   a) SeV Fmut: Sendai virus Strain Fushimi with NDV cleavage site    -   b) SeV Fmut dV: as a, in addition with mutations in the V and W        genes    -   c) SeV Fmut dC: as a, in addition with mutations in the C and C′        genes    -   d) SeV Fmut dCdY: as a, in addition with mutations in the C and        C′ genes and the Y1 and Y2 genes    -   e) SeV Fmut dCdV: as a, in addition with mutations in the C and        C′ genes, V and W genes    -   f) SeV Fmut dCdYdV: as a, in addition with mutations in the C        and C′ genes, V, W and Y1 and Y2 genes

8. Characterization of the Recombinant Sendai viruses

The generated recombinant Sendai viruses were extensively characterizedon several levels. The virus replication was analyzed on non-transformedVero producer cells and on tumor cells, in particular on hepatoma cells(Hep3B, HuH7, PLC/PRF/5), and on several non-tumor cells such as thehuman fibroblast cell line MRC 5 and primary human hepatocytes (PHH) ofvarious donors.

Growth curves

-   -   1. Provide 1×10⁵ cells per 12 well in 500 μl (Vero, hepatoma        cell, MRC 5, PHH are supplied, also starting from 1×10⁵ cells)    -   2. ON growth    -   3. Remove medium, 1× wash with PBS    -   4. +250 μl Optimem    -   5. Dilution of virus in Optimem (MOI 0.05)    -   6. Add diluted virus to the cells    -   7. Infect with all 6 SeV variants (freeze excess of the        dilutions and titrate as starting value)    -   8. Infect for 1 h at 37° C.    -   9. Wash cells 2×(PBS)    -   10. +1 ml fresh medium (DMEM 5% FCS)    -   11. Remove viruses in SN after 24, 48, 72 h, 96 h (in each case        after infection start) and store at −80° C. (200 μl each)        -   a. 2× careful wash with medium        -   b. +1000 ml medium (DMEM 5% FCS), scrap off cells    -   12. Freeze lysates at −80° C.    -   13. Thaw for titration        -   a. 2 min water bath 37° C.        -   b. 10-15 sec vortex        -   c. 2 min 3000×g small centrifuge    -   14. Centrifuge    -   15. Titration on Veros (TCID₅₀)        -   d. Prepare virus dilutions (first row always diluted)        -   e. Add to 96 well plates (always adjust fresh plate to 4°            C.)        -   f. Trypsinate cells        -   g. Count and add 2×10⁴ cells/96 well        -   h. Evaluate titer after 3 days

FIG. 9 shows the virus replication on Vero producer cells and hepatomacells. It is found that all generated recombinant viruses show a verygood replication in the Vero producer cells with comparable titers and avirus yield between 10⁷ and 10⁸ TCID₅₀/ml.

The titration of the virus particles was repeated in three independentassays, the mean value and the standard deviation are indicated(IO=inoculus).

In the three human standard cell lines for the hepatocellular carcinomaHuH7, PLC/PRF/5 and Hep3B it has been able to demonstrate that allrecombinant viruses show a very good replication in tumor cells.Therefore, one decisive precondition for a relevant oncolytic activityof the recombinant viruses according to the invention is fulfilled.

The human fibroblast cell line MRC-5 and primary human hepatocytes (PHH)of several different donors were exemplarily analyzed as non-tumorcells. The result is shown in FIG. 10. The intensity of the oncolysisfor the analyzed variants occurs in dependence of the deletions in theaccessory proteins. Viruses with a single deletion (SeV V F, mut dC, SeVV Fmut dV) have only moderately lost their capability to replicate innormal cells, whereas viruses with two or more deletions (SeV Fmut dCdY,SeV Fmut dCdV, SeV Fmut dCdYdV) replicate effectively in hepatoma cells,i.e. tumor cells, and destruct the latter, however infections in normalcells can hardly be observed.

The titration of the virus particles was repeated in three independentassays (for PHH with three different donors). Indicated are the meanvalue and the standard deviation (IO—inokulum).

In a further experiment the virus expansion in vivo was analyzed.

-   -   1. 5×10⁶ HuH7 tumor cells were subcutaneously implanted into        Balb c nu/nu mice under the right flank    -   2. Once a tumor volume of at least 100 mm³ has been reached the        Sendai virus was administered in 100 μl PBS into the tumor.    -   3. 2 days after the injection of the virus the animals were        sacrificed and the tumors were removed.    -   4. A part of the tumors was embedded into tissue tack, frozen        and cut by a cryotome. GFP expressed by the virus was directly        detected in the fluorescence microscope.    -   5. Another part of the tumors was embedded into paraffin and GFP        was detected by specific anti-GFP antibodies.

The result is shown in FIG. 11. It could have been demonstrated in vivothat the tumor cells are infected by the recombinant virus SeV Fmut andthe virus is in a position to expand therein. This result confirms thata replication in vivo outside the lung, as desired, in tumor tissue ispossible. SeV Fmut viruses are in a position to proliterate in humanhepatoma xenograft tissue (HuH7), D52 viruses (“Fushimi” wild typevariants) remain highly locally limited after the virus application intothe tumor.

9. Conclusion

The inventors were able to demonstrate by means of different recombinantSendai viruses which have been genetically modified in respect to thewild type in their F gene and their P gene that they can be used asoncolytic viruses in the anti-tumor therapy.

The following sequences are listed:

-   -   SEQ ID NO: 1: Nucleotide sequence of the F gene of SeV (Strain        “Ohita”)    -   SEQ ID NO: 2: Amino acid sequence of the F protein of SeV        (Strain “Ohita”)    -   SEQ ID NO: 3: Nucleotide sequence of the P gene of SeV (Strain        “Ohita”)    -   SEQ ID NO: 4: Amino acid sequence of the P protein of SeV        (Strain “Ohita”)    -   SEQ ID NO: 5 Amino acid sequence of the SeV-WT protease cleavage        site    -   SEQ ID NO: 6: Amino acid sequence of the protease cleavage site        of the F protein of NDV    -   SEQ ID NO: 7: Nucleotide sequence of the C′ gene of SeV (Strain        “Ohita”)    -   SEQ ID NO: 8: Amino acid sequence of the C′ protein of SeV        (Strain “Ohita”)    -   SEQ ID NO: 9: Nucleotide sequence of the C gene of SeV (Strain        “Ohita”)    -   SEQ ID NO: 10: Amino acid sequence of the C protein of SeV        (Strain “Ohita”)    -   SEQ ID NO: 11: Nucleotide sequence of the V gene of SeV (Strain        “Hamamatsu”)    -   SEQ ID NO: 12: Amino acid sequence of the V protein of SeV        (Strain “Hamamatsu”)    -   SEQ ID NO: 13: Nucleotide sequence of the W gene of SeV (Strain        “Cantel)”)    -   SEQ ID NO: 14: Amino acid sequence of the W protein of SeV        (Strain “Cantel)”)    -   SEQ ID NO: 15: Nucleotide sequence of the Y1 gene of SeV (Strain        “Ohita”)    -   SEQ ID NO: 16: Amino acid sequence of the Y1 protein of SeV        (Strain “Ohita”)    -   SEQ ID NO: 17: Nucleotide sequence of the Y2 gene of SeV (Strain        “52”)    -   SEQ ID NO: 18: Amino acid sequence of the Y2 protein of SeV        (Strain “52”)    -   SEQ ID NO: 19: Nucleotide sequence of the cloning region of        pSL1180    -   SEQ ID NO: 20: Nucleotide sequence PCR primer 1    -   SEQ ID NO: 21: Nucleotide sequence P gene    -   SEQ ID NO: 22: Nucleotide sequence P gene mut.    -   SEQ ID NO: 23: Nucleotide sequence C gene mut.    -   SEQ ID NO: 24: Nucleotide sequence PCR primer 2    -   SEQ ID NO: 25: Nucleotide sequence P gene    -   SEQ ID NO: 26: Nucleotide sequence Y1 gene    -   SEQ ID NO: 27: Nucleotide sequence Y1 gene mut.    -   SEQ ID NO: 28: Nucleotide sequence PCR primer 3    -   SEQ ID NO: 29: Nucleotide sequence P gene    -   SEQ ID NO: 30: Nucleotide sequence P gene mut.    -   SEQ ID NO: 31: Nucleotide sequence V gene mut.    -   SEQ ID NO: 32: Nucleotide sequence W gene mut.    -   SEQ ID NO: 33: Nucleotide sequence of the cleavage site in the F        gene of SeV    -   SEQ ID NO: 34: Nucleotide sequence of the cleavage site in the F        gene of NDV

What is claimed is:
 1. A genetically modified Paramyxovirus which, inreference to the wild type (wt), comprises in its F gene at least afirst genetic modification and in its P gene at least a second geneticmodification.
 2. The genetically modified Paramyxovirus of claim 1,wherein the first genetic modification results in a tropism extension.3. The genetically modified Paramyxovirus of claim 1, wherein the secondgenetic modification results in a tropism restriction.
 4. Thegenetically modified Paramyxovirus of claim 1, which is a geneticallymodified Sendai virus (SeV).
 5. The genetically modified Paramyxovirusof claim 4, wherein through the first genetic modification thenucleotide sequence encoding the SeV wt protease cleavage site isreplaced by a nucleotide sequence encoding a ubiquitous proteasecleavage site.
 6. The genetically modified Paramyxovirus of claim 5,wherein the ubiquitous protease cleavage site originates from the F geneof the Newcastle disease virus (NDV).
 7. The genetically modifiedParamyxovirus of claim 5, wherein the SeV wt protease cleavage sitecomprises the amino acid sequence VPQSR (SEQ ID no. 5) and theubiquitous protease cleavage site comprises the amino acid sequenceRRQKR (SEQ ID no. 6).
 8. The genetically modified Paramyxovirus of claim1, wherein through the second genetic modification the nucleotidesequence encoding at least one of the accessory non-structural proteinsencoded by the P gene is modified.
 9. The genetically modifiedParamyxovirus of claim 8, wherein through the second geneticmodification at least one of the accessory non-structural proteins isnon-transcribable.
 10. The genetically modified Paramyxovirus of claim9, wherein through the second genetic modification the start codon of atleast one of the accessory non-structural proteins is functionallydestroyed.
 11. The genetically modified Paramyxovirus of claim 8,wherein the accessory non-structural proteins are selected from thegroup consisting of: C′, C, Y1, Y2, V and W.
 12. The geneticallymodified Paramyxovirus of claim 11, wherein through the second geneticmodification at least the C(C or C′) and Y (Y1 or Y2) proteins arefunctionally destroyed.
 13. The genetically modified Paramyxovirus ofclaim 11, wherein the second genetic modification at least the C(C orC′), V and W proteins are functionally destroyed.
 14. The geneticallymodified Paramyxovirus of claim 11, wherein through the second geneticmodification at least the C(C or C′), V, W and Y (Y1 or Y2) proteins arefunctionally destroyed.
 15. The genetically modified Paramyxovirus ofclaim 1, which, in relation to the wild type (wt), comprises at leastone transgene.
 16. The genetically modified Paramyxovirus of claim 15,wherein the transgene is a suicide gene or another gene that inducescell death or an immunostimulating gene.
 17. A pharmaceuticalcomposition comprising the genetically modified Paramyxovirus of claim 1and a pharmaceutically acceptable carrier.
 18. A method for theproduction of a pharmaceutical composition for the therapeutical and/orprophylactical treatment of a tumor disease, comprising the followingsteps: (1) providing the genetically modified Paramyxovirus of claim 1,and (2) formulating the genetically modified Paramyxovirus into anacceptable carrier.